The goal of this project is to develop UDEP antibiotics, which cause bacterial cells to self-digest through activation of the ClpP protease. This unique ?activating? mechanism causes rapid and exceptional killing of drug resistant pathogens. UDEPs also have an important advantage ? killing of both metabolically active and dormant forms of pathogens. Many bacteria evade killing by traditional antibiotics, even surviving high concentrations for prolonged periods by simply growing slowly or not at all, in the case of persister cells. These surviving cells contribute to phenotypic antibiotic resistance, cause recurrent infections, and explain why traditional antibiotics are unable to kill biofilms, which have restricted access to the immune system. However, bacteria cannot escape death by shutting down or waiting until antibiotic levels drop upon activation of ClpP proteases by UDEPs. UDEPs not only target antimicrobial resistant pathogens, but may also allow common infections to be treated more quickly and effectively with less recurrence, while chronic infections like endocarditis, osteomyelitis, catheter-related bloodstream infections and prosthetic joint infections could be cured with antibiotics for the first time. A structure guided medicinal chemistry program led to the discovery of the UDEPs series, many of which have superior drug-like properties compared to the first generation acyldepsipeptides. Major gains in area under the curve (AUC), Cmax, half-life, and clearance have been achieved, while maintaining potency. A recent structural advance has enabled us to prioritize a lead-like UDEP, 3349. This compound displays efficacy in multiple animal models of infection which are highly predictive for humans, including septicemia, neutropenic thigh, pneumonia, and in a complicated model of biofilm foreign body infection. In this study, 3349 will be used to benchmark a sub-library of late leads designed to further improve druggability to produce a lead candidate suitable to be advanced into pre-clinical development. These studies will be performed in four aims: (i) Further optimization of late lead UDEPs using structure and PK guided design; (ii) in vitro pharmacological profiling to maximize safety, and hollow-fiber models of infection will be used to guide dose selections and the choice of antibiotic partners; (iii) in vivo efficacy determination in infection models of peritonitis septicemia, neutropenic thigh, lung pneumonia, implanted catheter biofilms and endocarditis; (iv) Preclinical development, using detailed in vivo PK/PD dose, dosing interval and target attainment will support IND-enabling studies. Toxicokinetic studies will support dose selection, toxicology endpoints and toxicokinetic time points for GLP-compliant studies. These studies will provide the basis for a risk- benefit assessment prior to meeting with the FDA.